本文選題：aroA基因 + 草甘膦抗性��； 參考：《華中農(nóng)業(yè)大學》2013年碩士論文

【摘要】：5-磷酸烯醇式丙酮酸-3-磷酸莽草酸合成酶(EPSPS)是草甘膦的靶標酶,轉基因抗草甘膦作物主要通過轉入外源EPSPS基因來培育。抗草甘膦基因(EPSPS)的克隆、表達及其功能驗證等是現(xiàn)代植物分子育種研究的重點。因此,加大挖掘相關基因資源和基因改造力度,對進一步獲取具有獨立知識產(chǎn)權的抗草甘膦新基因有重要意義。 本研究從華南地區(qū)的農(nóng)田和水體中采樣,并對樣品進行草甘膦處理,用含不同濃度梯度的草甘膦選擇性培養(yǎng)基分離得到1株草甘膦抗性細菌和3株抗性真菌。通過對抗性菌株的形態(tài)觀察和基于16S rDNA序列、ITS序列分析的系統(tǒng)發(fā)育分類方法,鑒定出該抗性細菌為肺炎克雷伯氏菌(Klebsiella pneumoniae),暫定名為kpS001；鑒定出3株抗性真菌分別為淡紫擬青霉(Paeciloomyces lilacinus)、多變根毛霉(Rhizomucor variabilis)和黃曲霉(Aspergillus flavus),分別命名為F35,F37,F62。 克隆出肺炎克雷伯氏菌kpS001的aroA基因,命名為aroAs001對其序列進行分析,發(fā)現(xiàn)aroAs001基因片段長度為1284bp,在氨基酸序列的91-98和第175-183位有EPSP酶功能保守序列-L-G-N-A-G-T-A-和-A-L-L-M-T-A-P-L-A-。將該氨基酸序列與NCBI中Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044全長aroA基因編碼的氨基酸序列相比,其編碼的氨基酸序列的第76位發(fā)生變異,由蘇氨酸突變?yōu)楫惲涟彼?推測該位點的變化可能是導致肺炎克雷伯氏菌kpS001菌株對草甘膦抗性增強的原因之一。 用pProA載體構建重組質粒pProA-aroAs001,轉入aroA缺陷性大腸桿菌菌株DH5a (DH5a/△aroA)得到目標重組菌。通過誘導該菌的蛋白表達,發(fā)現(xiàn)目的蛋白在重組菌中有特異表達,大小約為48kDa,與預期蛋白大小相符；然后將重組菌株接入到含不同濃度草甘膦的M9基礎鹽培養(yǎng)基中培養(yǎng),進行功能互補實驗。實驗結果顯示,轉入aroAs001基因的重組菌株的生長狀態(tài)明顯優(yōu)于對照組菌株；在含200mmol/L以下草甘膦的培養(yǎng)基中,重組菌的生長基本沒有受到影響,隨著草甘膦濃度增加,重組菌的生長逐漸受到抑制,但仍明顯高于對照菌株,重組菌能夠在350mmol/L草甘膦濃度的選擇性培養(yǎng)基上生長,表明克隆出來的aroA基因是kpS001對草甘膦產(chǎn)生抗性的主要原因。
[Abstract]:5-phosphoenolpyruvate-3-shikimic acid synthetase (EPSPS) is the target enzyme of glyphosate. The cloning, expression and functional verification of glyphosate resistant gene (EPSPS) are the focus of molecular breeding in modern plants. Therefore, it is of great significance to excavate related genetic resources and genetic modification to obtain new glyphosate resistant genes with independent intellectual property rights. In this study, glyphosate resistant bacteria and 3 resistant fungi were isolated from farmland and water in South China by glyphosate treatment. The resistant strain was identified as Klebsiella pneumoniae), as kpS001 by morphological observation and phylogenetic classification based on its sequence analysis of 16s rDNA sequence. Three strains of resistant fungi were identified as Paeciloomyces lilacinus), Rhizomucor variabilis and Aspergillus flavus (Aspergillus flavus), as F35, F37, F62, respectively. The aroA gene of Klebsiella pneumoniae kpS001 was cloned and named as aroAs001 to analyze its sequence. It was found that the length of aroAs001 gene was 1284 bp. there were EPSP functional conserved sequences -L-G-N-A-G-T-A- and -A-L-L-M-T-A-P-A-at the 91-98 and 175-183 amino acid sequences of Klebsiella pneumoniae. Compared with the amino acid sequence encoded by Klebsiella pneumoniae subsp. pneumoniae NTUH-K2044 full-length aroA gene, the amino acid sequence was mutated from threonine to isoleucine. It is speculated that the change of this site may be one of the reasons for the increase of resistance to glyphosate of Klebsiella pneumoniae kpS001. The recombinant plasmid pProA-aroAs001 was constructed by using pProA vector and transferred into aroA deficient Escherichia coli strain DH 5a (DH 5a / aroA) to obtain the target recombinant strain. By inducing the protein expression of the strain, we found that the target protein was specifically expressed in the recombinant strain, the size of which was about 48 kDa, which was in line with the expected protein size, and then the recombinant strain was transferred into the M9 basic salt medium containing different concentrations of glyphosate, and was cultured in the M9 basic salt medium containing different concentrations of glyphosate. The functional complementary experiment was carried out. The results showed that the growth state of the recombinant strain transferred to aroAs001 gene was significantly better than that of the control strain, and the growth of the recombinant strain was almost unaffected in the medium containing glyphosate below 200 mmol / L, with the increase of glyphosate concentration. The growth of the recombinant strain was inhibited gradually, but still significantly higher than that of the control strain. The recombinant strain could grow on the selective medium with 350 mmol / L glyphosate concentration, indicating that the cloned aroA gene was the main cause of the resistance of kpS001 to glyphosate.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2013
【分類號】：Q78

【參考文獻】

相關碩士學位論文 前1條

1 潘渠;草甘膦抗性和降解微生物的分離鑒定及生物學特性研究[D];四川大學;2001年

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本文編號：2106042